Multi-OMIC analysis of Huntington disease reveals a neuroprotective astrocyte state

Abstract

Huntington disease (HD) is an incurable neurodegenerative disease characterized by neuronal loss and astrogliosis. One hallmark of HD is the selective neuronal vulnerability of striatal medium spiny neurons. To date, the underlying mechanisms of this selective vulnerability have not been fully defined. Here, we employed a multi-omic approach including single nucleus RNAseq (snRNAseq), bulk RNAseq, lipidomics,HTTgene CAG repeat length measurements, and multiplexed immunofluorescence on post-mortem brain tissue from multiple brain regions of HD and control donors. We defined a signature of genes that is driven by CAG repeat length and found it enriched in astrocytic and microglial genes. Moreover, weighted gene correlation network analysis showed loss of connectivity of astrocytic and microglial modules in HD and identified modules that correlated with CAG-repeat length which further implicated inflammatory pathways and metabolism. We performed lipidomic analysis of HD and control brains and identified several lipid species that correlate with HD grade, including ceramides and very long chain fatty acids. Integration of lipidomics and bulk transcriptomics identified a consensus gene signature that correlates with HD grade and HD lipidomic abnormalities and implicated the unfolded protein response pathway. Because astrocytes are critical for brain lipid metabolism and play important roles in regulating inflammation, we analyzed our snRNAseq dataset with an emphasis on astrocyte pathology. We found two main astrocyte types that spanned multiple brain regions; these types correspond to protoplasmic astrocytes, and fibrous-like - CD44-positive, astrocytes. HD pathology was differentially associated with these cell types in a region-specific manner. One protoplasmic astrocyte cluster showed high expression of metallothionein genes, the depletion of this cluster positively correlated with the depletion of vulnerable medium spiny neurons in the caudate nucleus. We confirmed that metallothioneins were increased in cingulate HD astrocytes but were unchanged or even decreased in caudate astrocytes. We combined existing genome-wide association studies (GWAS) with a GWA study conducted on HD patients from the original Venezuelan cohort and identified a single-nucleotide polymorphism in the metallothionein gene locus associated with delayed age of onset. Functional studies found that metallothionein overexpressing astrocytes are better able to buffer glutamate and were neuroprotective of patient-derived directly reprogrammed HD MSNs as well as against rotenone-induced neuronal deathin vitro. Finally, we found that metallothionein-overexpressing astrocytes increased the phagocytic activity of microgliain vitroand increased the expression of genes involved in fatty acid binding. Together, we identified an astrocytic phenotype that is regionally-enriched in less vulnerable brain regions that can be leveraged to protect neurons in HD.

The spatial landscape of glial pathology and T-cell response in Parkinson’s disease substantia nigra

“…In addition to the increase of clonal T-cells in PD SN, we observed a transition from protoplasmic astrocytic phenotypes to a fibrous-like phenotype, which was coupled with a decrease in the neuroprotective protein MT3. This is similar to our previous findings in HD 36 , and highlights a region-specific response of astrocytes to neurodegeneration, which may result from, or be more likely to contribute to, neurodegeneration. Additional studies are needed to determine the generalizability of this phenomenon, and whether it is a root cause or a result of neurodegeneration.…”

“…S4a-b for cingulate cortex). Following our previous results that indicated a compensatory neuroprotective astrocytic response characterized by increased metallothionein protein MT3 expression in HD astrocytes 38,45 , we found that MT3 was increased in the CD44- (protoplasmic) cingulate cortex astrocytes but not SN protoplasmic astrocytes. As expected, expression of GFAP was increased in both regions ( Fig.…”

“…Interestingly, astrocytes showed increased MT3 expression in the cingulate in PD (Fig. 2d), as we have shown for Huntington’s disease (HD) 36 . We validated this finding in our supplemental IHC data ( Fig.…”

“…We then examined evidence for a transition from protoplasmic to fibrous-like states in PD, as we have seen in HD, hypoxia, and seizures 36,100 , using pseudotime analysis which is a means to order cells along a trajectory of gene signature. We ordered cells on the axis of variation along the genes that differ between the two astrocyte sublineages ( Fig.…”

“…Cingulate cortex neurons were assigned in line with Paryani et. al 2023 36 . Nuclei that did not conform to cell types were presumed to be doublets or artifactual noise and removed.…”